Molecular Organization and Function of Paranodal Axo-Glial Junctions The paranodal region of myelinated axons contains a specialized domain consisting of septate junctions formed between myelin loops and the axolemma. These axo-glial septate junctions (AGSJs) have been the object of considerable investigation. Their major molecular constituents are: Contactin-associated protein (CASPR), Contactin (CONT) and a 155kDa glial-isoform of Neurofascin (NF155). Genetic ablation of CASPR in mice results in the disruption of AGSJs, disorganization of the paranodal axonal cytoskeleton, and degeneration of the Purkinje axons. CONT and NF null mice also fail to form AGSJs. In further studies, we have created myelinating glial-specific NF155 mutant mice, which fail to cluster CASPR at the paranodes and also fail to form AGSJs. In addition, we have identified a PDZ domain-containing cytoskeletal protein that interacts with the cytoplasmic region of CASPR and localizes at the paranodes. Despite increasingly detailed knowledge of the AGSJs, there remain fundamental questions concerning their organization and their essential role in maintaining axonal health and neuronal function. In this proposal, we propose to use a combination of genetic, cell biological, molecular and biochemical methods to define the molecular relationship between AGSJs and axonal/glial cytoskeleton using mouse as a genetic model system. Our specific aims are: 1) Determine the role of glial NF155 in the organization and maintenance of the AGSJs. 2) Determine the structure/function relationship between NF155 and the glial cytoskeleton in the formation and/or stabilization of the AGSJs. 3) Determine the structure/function relationship between CASPR at the paranodal axolemma and axonal cytoskeletal components in the organization of the AGSJs. Collectively, the proposed studies should provide new and fundamental information that will bear directly on the organization of AGSJs and the mechanisms by which they function at the paranodes. In the future, these studies will advance our understanding of how AGSJs are compromised in myelin-related pathologies, like Multiple Sclerosis (MS). Our findings will also provide insights into the functional deficits that accompany MS at the paranodal axon-glial interface and help design pharmaceutical interventions to preserve the delicate relationship between the axons and the myelin-forming glial cells. Molecular Organization and Function of Paranodal Axo-Glial Junctions The studies described in this application relate to the molecular mechanisms that govern the establishment and organization of distinct axonal domains in the myelinated nerve fibers. This unique structure allows the saltatory propagation of the nerve impulses in the myelinated axons. Better understanding of these mechanisms may help to design future therapeutic strategies to myelin-related diseases or demyelination disorders like for example multiple sclerosis (MS) where remyelination is required and the axonal domain structure must be preserved.